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1.
Nat Cell Biol ; 23(1): 40-48, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33420492

RESUMO

Loss of the fragile X protein FMRP is a leading cause of intellectual disability and autism1,2, but the underlying mechanism remains poorly understood. We report that FMRP deficiency results in hyperactivated nonsense-mediated mRNA decay (NMD)3,4 in human SH-SY5Y neuroblastoma cells and fragile X syndrome (FXS) fibroblast-derived induced pluripotent stem cells (iPSCs). We examined the underlying mechanism and found that the key NMD factor UPF1 binds directly to FMRP, promoting FMRP binding to NMD targets. Our data indicate that FMRP acts as an NMD repressor. In the absence of FMRP, NMD targets are relieved from FMRP-mediated translational repression so that their half-lives are decreased and, for those NMD targets encoding NMD factors, increased translation produces abnormally high factor levels despite their hyperactivated NMD. Transcriptome-wide alterations caused by NMD hyperactivation have a role in the FXS phenotype. Consistent with this, small-molecule-mediated inhibition of hyperactivated NMD, which typifies iPSCs derived from patients with FXS, restores a number of neurodifferentiation markers, including those not deriving from NMD targets. Our mechanistic studies reveal that many molecular abnormalities in FMRP-deficient cells are attributable-either directly or indirectly-to misregulated NMD.


Assuntos
Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/patologia , Deleção de Genes , Neuroblastoma/patologia , Degradação do RNAm Mediada por Códon sem Sentido , Transcriptoma , Estudos de Casos e Controles , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/patologia , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neurônios/metabolismo , Neurônios/patologia , RNA-Seq , Transativadores
2.
Nucleic Acids Res ; 49(2): 1133-1151, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33406240

RESUMO

Alternative splicing generates multiple transcript and protein isoforms from a single gene and controls transcript intracellular localization and stability by coupling to mRNA export and nonsense-mediated mRNA decay (NMD). RNA interference (RNAi) is a potent mechanism to modulate gene expression. However, its interactions with alternative splicing are poorly understood. We used artificial microRNAs (amiRNAs, also termed shRNAmiR) to knockdown all splice variants of selected target genes in Arabidopsis thaliana. We found that splice variants, which vary by their protein-coding capacity, subcellular localization and sensitivity to NMD, are affected differentially by an amiRNA, although all of them contain the target site. Particular transcript isoforms escape amiRNA-mediated degradation due to their nuclear localization. The nuclear and NMD-sensitive isoforms mask RNAi action in alternatively spliced genes. Interestingly, Arabidopsis SPL genes, which undergo alternative splicing and are targets of miR156, are regulated in the same manner. Moreover, similar results were obtained in mammalian cells using siRNAs, indicating cross-kingdom conservation of these interactions among RNAi and splicing isoforms. Furthermore, we report that amiRNA can trigger artificial alternative splicing, thus expanding the RNAi functional repertoire. Our findings unveil novel interactions between different post-transcriptional processes in defining transcript fates and regulating gene expression.


Assuntos
Processamento Alternativo/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Técnicas de Silenciamento de Genes , Degradação do RNAm Mediada por Códon sem Sentido , Isoformas de Proteínas/genética , Interferência de RNA , Precursores de RNA/metabolismo , RNA de Plantas/metabolismo , Proteínas de Arabidopsis/biossíntese , Éxons , Genes de Plantas , Células HeLa , Humanos , MicroRNAs/genética , Plantas Geneticamente Modificadas , Isoformas de Proteínas/biossíntese , Protoplastos/metabolismo , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , RNA de Plantas/genética , Fatores de Processamento de Serina-Arginina/biossíntese , Fatores de Processamento de Serina-Arginina/genética , Transcrição Genética , Transfecção
3.
Int J Mol Sci ; 21(24)2020 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-33321981

RESUMO

Alternative splicing (AS) of precursor mRNA (pre-mRNA) is a cellular post-transcriptional process that generates protein isoform diversity. Nonsense-mediated RNA decay (NMD) is an mRNA surveillance pathway that recognizes and selectively degrades transcripts containing premature translation-termination codons (PTCs), thereby preventing the production of truncated proteins. Nevertheless, NMD also fine-tunes the gene expression of physiological mRNAs encoding full-length proteins. Interestingly, around one third of all AS events results in PTC-containing transcripts that undergo NMD. Numerous studies have reported a coordinated action between AS and NMD, in order to regulate the expression of several genes, especially those coding for RNA-binding proteins (RBPs). This coupling of AS to NMD (AS-NMD) is considered a gene expression tool that controls the ratio of productive to unproductive mRNA isoforms, ultimately degrading PTC-containing non-functional mRNAs. In this review, we focus on the mechanisms underlying AS-NMD, and how this regulatory process is able to control the homeostatic expression of numerous RBPs, including splicing factors, through auto- and cross-regulatory feedback loops. Furthermore, we discuss the importance of AS-NMD in the regulation of biological processes, such as cell differentiation. Finally, we analyze interesting recent data on the relevance of AS-NMD to human health, covering its potential roles in cancer and other disorders.


Assuntos
Processamento Alternativo , Degradação do RNAm Mediada por Códon sem Sentido , Animais , Regulação da Expressão Gênica no Desenvolvimento , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
4.
Int J Mol Sci ; 21(24)2020 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-33348896

RESUMO

SF3B1 is a core component of the U2 spliceosome that is frequently mutated in cancer. We have previously shown that titrating the activity of SF3B1, using the inhibitor pladienolide B (PB), affects distinct steps of the heat shock response (HSR). Here, we identify other genes that are sensitive to different levels of SF3B1 (5 vs. 100 nM PB) using RNA sequencing. Significant changes to mRNA splicing were identified at both low PB and high PB concentrations. Changes in expression were also identified in the absence of alternative splicing, suggesting that SF3B1 influences other gene expression pathways. Surprisingly, gene expression changes identified in low PB are not predictive of changes in high PB. Specific pathways were identified with differential sensitivity to PB concentration, including nonsense-mediated decay and protein-folding homeostasis, both of which were validated using independent reporter constructs. Strikingly, cells exposed to low PB displayed enhanced protein-folding capacity relative to untreated cells. These data reveal that the transcriptome is exquisitely sensitive to SF3B1 and suggests that the activity of SF3B1 is finely regulated to coordinate mRNA splicing, gene expression and cellular physiology.


Assuntos
Processamento Alternativo , Fenômenos Fisiológicos Celulares , Regulação da Expressão Gênica/efeitos dos fármacos , Fosfoproteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma/efeitos dos fármacos , Compostos de Epóxi/farmacologia , Células HEK293 , Humanos , Macrolídeos/farmacologia , Degradação do RNAm Mediada por Códon sem Sentido , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , RNA Mensageiro/genética
5.
Nucleic Acids Res ; 48(18): 10259-10279, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32941650

RESUMO

To gain insight into the mechanistic link between translation termination and nonsense-mediated mRNA decay (NMD), we depleted the ribosome recycling factor ABCE1 in human cells, resulting in an upregulation of NMD-sensitive mRNAs. Suppression of NMD on these mRNAs occurs prior to their SMG6-mediated endonucleolytic cleavage. ABCE1 depletion caused ribosome stalling at termination codons (TCs) and increased ribosome occupancy in 3' UTRs, implying enhanced TC readthrough. ABCE1 knockdown indeed increased the rate of readthrough and continuation of translation in different reading frames, providing a possible explanation for the observed NMD inhibition, since enhanced readthrough displaces NMD activating proteins from the 3' UTR. Our results indicate that stalling at TCs triggers ribosome collisions and activates ribosome quality control. Collectively, we show that improper translation termination can lead to readthrough of the TC, presumably due to ribosome collisions pushing the stalled ribosomes into the 3' UTR, where it can resume translation in-frame as well as out-of-frame.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Códon de Terminação/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Telomerase/genética , Regiões 3' não Traduzidas/genética , Mudança da Fase de Leitura do Gene Ribossômico/genética , Humanos , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Ribossomos/genética
6.
Nat Commun ; 11(1): 4134, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807779

RESUMO

Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway that is important for the elimination of faulty, and the regulation of normal, mRNAs. The molecular details of the early steps in NMD are not fully understood but previous work suggests that NMD activation occurs as a consequence of ribosome stalling at the termination codon (TC). To test this hypothesis, we established an in vitro translation-coupled toeprinting assay based on lysates from human cells that allows monitoring of ribosome occupancy at the TC of reporter mRNAs. In contrast to the prevailing NMD model, our in vitro system reveals similar ribosomal occupancy at the stop codons of NMD-sensitive and NMD-insensitive reporter mRNAs. Moreover, ribosome profiling reveals a similar density of ribosomes at the TC of endogenous NMD-sensitive and NMD-insensitive mRNAs in vivo. Together, these data show that NMD activation is not accompanied by stable stalling of ribosomes at TCs.


Assuntos
Degradação do RNAm Mediada por Códon sem Sentido/fisiologia , Ribossomos/metabolismo , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/fisiologia , Códon de Terminação/genética , Humanos , Degradação do RNAm Mediada por Códon sem Sentido/genética , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/genética
7.
PLoS Pathog ; 16(8): e1008346, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32764824

RESUMO

Viruses subvert macromolecular pathways in infected host cells to aid in viral gene amplification or to counteract innate immune responses. Roles for host-encoded, noncoding RNAs, including microRNAs, have been found to provide pro- and anti-viral functions. Recently, circular RNAs (circRNAs), that are generated by a nuclear back-splicing mechanism of pre-mRNAs, have been implicated to have roles in DNA virus-infected cells. This study examines the circular RNA landscape in uninfected and hepatitis C virus (HCV)-infected liver cells. Results showed that the abundances of distinct classes of circRNAs were up-regulated or down-regulated in infected cells. Identified circRNAs displayed pro-viral effects. One particular up-regulated circRNA, circPSD3, displayed a very pronounced effect on viral RNA abundances in both hepatitis C virus- and Dengue virus-infected cells. Though circPSD3 has been shown to bind factor eIF4A3 that modulates the cellular nonsense-mediated decay (NMD) pathway, circPSD3 regulates RNA amplification in a pro-viral manner at a post-translational step, while eIF4A3 exhibits the anti-viral property of the NMD pathway. Findings from the global analyses of the circular RNA landscape argue that pro-, and likely, anti-viral functions are executed by circRNAs that modulate viral gene expression as well as host pathways. Because of their long half-lives, circRNAs likely play hitherto unknown, important roles in viral pathogenesis.


Assuntos
Carcinoma Hepatocelular/virologia , Hepacivirus/genética , Hepatite C/complicações , Neoplasias Hepáticas/virologia , Provírus/genética , RNA Circular/genética , RNA Viral/genética , Replicação Viral , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Perfilação da Expressão Gênica , Hepatite C/virologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Degradação do RNAm Mediada por Códon sem Sentido , Proteínas Virais/genética , Proteínas Virais/metabolismo
9.
Nat Commun ; 11(1): 3800, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32733040

RESUMO

Frameshift insertion/deletions (fs-indels) are an infrequent but highly immunogenic mutation subtype. Although fs-indels are degraded through the nonsense-mediated decay (NMD) pathway, we hypothesise that some fs-indels escape degradation and elicit anti-tumor immune responses. Using allele-specific expression analysis, expressed fs-indels are enriched in genomic positions predicted to escape NMD, and associated with higher protein expression, consistent with degradation escape (NMD-escape). Across four independent melanoma cohorts, NMD-escape mutations are significantly associated with clinical-benefit to checkpoint inhibitor (CPI) therapy (Pmeta = 0.0039). NMD-escape mutations are additionally found to associate with clinical-benefit in the low-TMB setting. Furthermore, in an adoptive cell therapy treated melanoma cohort, NMD-escape mutation count is the most significant biomarker associated with clinical-benefit. Analysis of functional T cell reactivity screens from personalized vaccine studies shows direct evidence of fs-indel derived neoantigens eliciting immune response, particularly those with highly elongated neo open reading frames. NMD-escape fs-indels represent an attractive target for biomarker optimisation and immunotherapy design.


Assuntos
Melanoma/genética , Melanoma/imunologia , Degradação do RNAm Mediada por Códon sem Sentido/genética , Linfócitos T/imunologia , Evasão Tumoral/genética , Transferência Adotiva , Antígenos de Neoplasias/imunologia , Biomarcadores Tumorais/genética , Mutação da Fase de Leitura/genética , Humanos , Mutação INDEL/genética , Imunoterapia Adotiva , Linfócitos T/transplante , Sequenciamento Completo do Exoma
10.
Proc Natl Acad Sci U S A ; 117(36): 22390-22401, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32848063

RESUMO

Targeted cancer therapy aims to achieve specific elimination of cancerous but not normal cells. Recently, PIWI proteins, a subfamily of the PAZ-PIWI domain (PPD) protein family, have emerged as promising candidates for targeted cancer therapy. PPD proteins are essential for small noncoding RNA pathways. The Argonaute subfamily partners with microRNA and small interfering RNA, whereas the PIWI subfamily partners with PIWI-interacting RNA (piRNA). Both PIWI proteins and piRNA are mostly expressed in the germline and best known for their function in transposon silencing, with no detectable function in mammalian somatic tissues. However, PIWI proteins become aberrantly expressed in multiple types of somatic cancers, thus gaining interest in targeted therapy. Despite this, little is known about the regulatory mechanism of PIWI proteins in cancer. Here we report that one of the four PIWI proteins in humans, PIWIL1, is highly expressed in gastric cancer tissues and cell lines. Knocking out the PIWIL1 gene (PIWIL1-KO) drastically reduces gastric cancer cell proliferation, migration, metastasis, and tumorigenesis. RNA deep sequencing of gastric cancer cell line SNU-1 reveals that KO significantly changes the transcriptome, causing the up-regulation of most of its associated transcripts. Surprisingly, few bona fide piRNAs exist in gastric cancer cells. Furthermore, abolishing the piRNA-binding activity of PIWIL1 does not affect its oncogenic function. Thus, PIWIL1 function in gastric cancer cells is independent of piRNA. This piRNA-independent regulation involves interaction with the UPF1-mediated nonsense-mediated mRNA decay (NMD) mechanism. Altogether, our findings reveal a piRNA-independent function of PIWIL1 in promoting gastric cancer.


Assuntos
Proteínas Argonauta , RNA Interferente Pequeno , Neoplasias Gástricas , Animais , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Linhagem Celular Tumoral , Feminino , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Camundongos Nus , Degradação do RNAm Mediada por Códon sem Sentido/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Estômago/química , Estômago/patologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia
11.
Neuron ; 107(6): 1180-1196.e8, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32710818

RESUMO

Continuous neuronal survival is vital for mammals because mammalian brains have limited regeneration capability. After neurogenesis, suppression of apoptosis is needed to ensure a neuron's long-term survival. Here we describe a robust genetic program that intrinsically attenuates apoptosis competence in neurons. Developmental downregulation of the splicing regulator PTBP1 in immature neurons allows neural-specific splicing of the evolutionarily conserved Bak1 microexon 5. Exon 5 inclusion triggers nonsense-mediated mRNA decay (NMD) and unproductive translation of Bak1 transcripts (N-Bak mRNA), leading to suppression of pro-apoptotic BAK1 proteins and allowing neurons to reduce apoptosis. Germline heterozygous ablation of exon 5 increases BAK1 proteins exclusively in the brain, inflates neuronal apoptosis, and leads to early postnatal mortality. Therefore, neural-specific exon 5 splicing and depletion of BAK1 proteins uniquely repress neuronal apoptosis. Although apoptosis is important for development, attenuation of apoptosis competence through neural-specific splicing of the Bak1 microexon is essential for neuronal and animal survival.


Assuntos
Apoptose , Neurogênese , Processamento de RNA , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo
12.
Wiley Interdiscip Rev RNA ; 11(5): e1614, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32638509

RESUMO

Coronaviruses, including SARS-Cov-2, are RNA-based pathogens that interface with a large variety of RNA-related cellular processes during infection. These processes include capping, polyadenylation, localization, RNA stability, translation, and regulation by RNA binding proteins or noncoding RNA effectors. The goal of this article is to provide an in-depth perspective on the current state of knowledge of how various coronaviruses interact with, usurp, and/or avoid aspects of these cellular RNA biology machineries. A thorough understanding of how coronaviruses interact with RNA-related posttranscriptional processes in the cell should allow for new insights into aspects of viral pathogenesis as well as identify new potential avenues for the development of anti-coronaviral therapeutics. This article is categorized under: RNA in Disease and Development > RNA in Disease.


Assuntos
Betacoronavirus/genética , Interações Hospedeiro-Patógeno/genética , MicroRNAs/genética , RNA Circular/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Viral/genética , Animais , Betacoronavirus/metabolismo , Humanos , MicroRNAs/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , Poliadenilação , Biossíntese de Proteínas , Edição de RNA , Processamento de RNA , Estabilidade de RNA , RNA Circular/metabolismo , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Vírus da SARS/genética , Vírus da SARS/metabolismo
13.
Proc Natl Acad Sci U S A ; 117(28): 16456-16464, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32616572

RESUMO

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene leading to the presence of premature termination codons (PTC). Previous transcriptional studies have shown reduced DMD transcript levels in DMD patient and animal model muscles when PTC are present. Nonsense-mediated decay (NMD) has been suggested to be responsible for the observed reduction, but there is no experimental evidence supporting this claim. In this study, we aimed to investigate the mechanism responsible for the drop in DMD expression levels in the presence of PTC. We observed that the inhibition of NMD does not normalize DMD gene expression in DMD. Additionally, in situ hybridization showed that DMD messenger RNA primarily localizes in the nuclear compartment, confirming that a cytoplasmic mechanism like NMD indeed cannot be responsible for the observed reduction. Sequencing of nascent RNA to explore DMD transcription dynamics revealed a lower rate of DMD transcription in patient-derived myotubes compared to healthy controls, suggesting a transcriptional mechanism involved in reduced DMD transcript levels. Chromatin immunoprecipitation in muscle showed increased levels of the repressive histone mark H3K9me3 in mdx mice compared to wild-type mice, indicating a chromatin conformation less prone to transcription in mdx mice. In line with this finding, treatment with the histone deacetylase inhibitor givinostat caused a significant increase in DMD transcript expression in mdx mice. Overall, our findings show that transcription dynamics across the DMD locus are affected by the presence of PTC, hinting at a possible epigenetic mechanism responsible for this process.


Assuntos
Códon sem Sentido/genética , Distrofina/genética , Distrofia Muscular de Duchenne/genética , RNA Mensageiro/genética , Animais , Códon sem Sentido/metabolismo , Modelos Animais de Doenças , Distrofina/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , RNA Mensageiro/metabolismo
14.
Nat Commun ; 11(1): 3354, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620797

RESUMO

Expansion of an intronic (GGGGCC)n repeat region within the C9orf72 gene is a main cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). A hallmark of c9ALS/FTD is the accumulation of misprocessed RNAs, which are often targets of cellular RNA surveillance. Here, we show that RNA decay mechanisms involving upstream frameshift 1 (UPF1), including nonsense-mediated decay (NMD), are inhibited in c9ALS/FTD brains and in cultured cells expressing either of two arginine-rich dipeptide repeats (R-DPRs), poly(GR) and poly(PR). Mechanistically, although R-DPRs cause the recruitment of UPF1 to stress granules, stress granule formation is independent of NMD inhibition. Instead, NMD inhibition is primarily a result from global translational repression caused by R-DPRs. Overexpression of UPF1, but none of its NMD-deficient mutants, enhanced the survival of neurons treated by R-DPRs, suggesting that R-DPRs cause neurotoxicity in part by inhibiting cellular RNA surveillance.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteína C9orf72/genética , Demência Frontotemporal/genética , Degradação do RNAm Mediada por Códon sem Sentido , RNA Helicases/metabolismo , Transativadores/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Expansão das Repetições de DNA , Conjuntos de Dados como Assunto , Embrião de Mamíferos , Feminino , Lobo Frontal/patologia , Demência Frontotemporal/patologia , Humanos , Íntrons/genética , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Cultura Primária de Células , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA-Seq , Transativadores/genética
15.
Nat Commun ; 11(1): 3345, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620802

RESUMO

Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved RNA decay mechanism that has emerged as a potent cell-intrinsic restriction mechanism of retroviruses and positive-strand RNA viruses. However, whether NMD is capable of restricting DNA viruses is not known. The DNA virus Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma and primary effusion lymphoma (PEL). Here, we demonstrate that NMD restricts KSHV lytic reactivation. Leveraging high-throughput transcriptomics we identify NMD targets transcriptome-wide in PEL cells and identify host and viral RNAs as substrates. Moreover, we identified an NMD-regulated link between activation of the unfolded protein response and transcriptional activation of the main KSHV transcription factor RTA, itself an NMD target. Collectively, our study describes an intricate relationship between cellular targets of an RNA quality control pathway and KSHV lytic gene expression, and demonstrates that NMD can function as a cell intrinsic restriction mechanism acting upon DNA viruses.


Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/genética , Degradação do RNAm Mediada por Códon sem Sentido , RNA Viral/metabolismo , Ativação Viral/genética , Linhagem Celular Tumoral , Células HEK293 , Herpesvirus Humano 8/metabolismo , Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/virologia , RNA Mensageiro/metabolismo , RNA-Seq , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/virologia , Transativadores/genética , Transativadores/metabolismo , Ativação Transcricional , Resposta a Proteínas não Dobradas/genética , Latência Viral/genética
16.
Nucleic Acids Res ; 48(15): 8626-8644, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32621609

RESUMO

The exon junction complex (EJC) is an essential constituent and regulator of spliced messenger ribonucleoprotein particles (mRNPs) in metazoans. As a core component of the EJC, CASC3 was described to be pivotal for EJC-dependent nuclear and cytoplasmic processes. However, recent evidence suggests that CASC3 functions differently from other EJC core proteins. Here, we have established human CASC3 knockout cell lines to elucidate the cellular role of CASC3. In the knockout cells, overall EJC composition and EJC-dependent splicing are unchanged. A transcriptome-wide analysis reveals that hundreds of mRNA isoforms targeted by nonsense-mediated decay (NMD) are upregulated. Mechanistically, recruiting CASC3 to reporter mRNAs by direct tethering or via binding to the EJC stimulates mRNA decay and endonucleolytic cleavage at the termination codon. Building on existing EJC-NMD models, we propose that CASC3 equips the EJC with the persisting ability to communicate with the NMD machinery in the cytoplasm. Collectively, our results characterize CASC3 as a peripheral EJC protein that tailors the transcriptome by promoting the degradation of EJC-dependent NMD substrates.


Assuntos
Proteínas de Neoplasias/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Processamento de RNA/genética , Proteínas de Ligação a RNA/genética , Transcriptoma/genética , Sequência de Aminoácidos/genética , Núcleo Celular/genética , Éxons/genética , Técnicas de Inativação de Genes , Humanos , RNA Mensageiro/genética , Ribonucleoproteínas/genética
17.
RNA ; 26(11): 1509-1518, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32699064

RESUMO

Viruses have evolved in tandem with the organisms that they infect. Afflictions of the plant and animal kingdoms with viral infections have forced the host organism to evolve new or exploit existing systems to develop the countermeasures needed to offset viral insults. As one example, nonsense-mediated mRNA decay, a cellular quality-control mechanism ensuring the translational fidelity of mRNA transcripts, has been used to restrict virus replication in both plants and animals. In response, viruses have developed a slew of means to disrupt or become insensitive to NMD, providing researchers with potential new reagents that can be used to more fully understand the NMD mechanism.


Assuntos
Interações Hospedeiro-Patógeno , Degradação do RNAm Mediada por Códon sem Sentido , Vírus/metabolismo , Animais , Humanos , Plantas/virologia , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas Virais/genética , Fenômenos Fisiológicos Virais , Vírus/classificação , Vírus/genética , Vírus/crescimento & desenvolvimento
18.
Nat Commun ; 11(1): 3106, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561765

RESUMO

Nonsense-mediated mRNA decay (NMD) typifies an mRNA surveillance pathway. Because NMD necessitates a translation event to recognize a premature termination codon on mRNAs, truncated misfolded polypeptides (NMD-polypeptides) could potentially be generated from NMD substrates as byproducts. Here, we show that when the ubiquitin-proteasome system is overwhelmed, various misfolded polypeptides including NMD-polypeptides accumulate in the aggresome: a perinuclear nonmembranous compartment eventually cleared by autophagy. Hyperphosphorylation of the key NMD factor UPF1 is required for selective targeting of the misfolded polypeptide aggregates toward the aggresome via the CTIF-eEF1A1-DCTN1 complex: the aggresome-targeting cellular machinery. Visualization at a single-particle level reveals that UPF1 increases the frequency and fidelity of movement of CTIF aggregates toward the aggresome. Furthermore, the apoptosis induced by proteotoxic stresses is suppressed by UPF1 hyperphosphorylation. Altogether, our data provide evidence that UPF1 functions in the regulation of a protein surveillance as well as an mRNA quality control.


Assuntos
Degradação do RNAm Mediada por Códon sem Sentido , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Transativadores/metabolismo , Resposta a Proteínas não Dobradas/genética , Autofagia , Códon sem Sentido , Complexo Dinactina/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Células HEK293 , Células HeLa , Humanos , Imagem Molecular , Fator 1 de Elongação de Peptídeos/metabolismo , Fosforilação , Agregados Proteicos/genética , Imagem Individual de Molécula , Ubiquitina/metabolismo
19.
Proc Natl Acad Sci U S A ; 117(27): 15799-15808, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571908

RESUMO

The transcriptome of eukaryotic cells is constantly monitored for errors to avoid the production of undesired protein variants. The evolutionarily conserved nonsense-mediated mRNA decay (NMD) pathway degrades aberrant mRNAs, but also functions in the regulation of transcript abundance in response to changed physiological states. Here, we describe a zebrafish mutant of upf1, encoding the central component of the NMD machinery. Fish homozygous for the upf1 t20450 allele (Y163X) survive until day 10 after fertilization, presenting with impaired T cell development as one of the most conspicuous features of the mutant phenotype. Analysis of differentially expressed genes identified dysregulation of the pre-mRNA splicing pathway, accompanied by perturbed autoregulation of canonical splicing activators (SRSF) and repressors (HNRNP). In upf1-deficient mutants, NMD-susceptible transcripts of ribosomal proteins that are known for their role as noncanonical splicing regulators were greatly increased, most notably, rpl10a When the levels of NMD-susceptible rpl10a transcripts were artificially increased in zebrafish larvae, T cell development was significantly impaired, suggesting that perturbed autoregulation of rpl10a splicing contributes to failing T cell development in upf1 deficiency. Our results identify an extraribosomal tissue-specific function to rpl10a in the immune system, and thus exemplify the advantages of the zebrafish model to study the effects of upf1-deficiency in the context of a vertebrate organism.


Assuntos
Glutationa/análogos & derivados , Degradação do RNAm Mediada por Códon sem Sentido/genética , Processamento de RNA/genética , Proteínas de Ligação a RNA/genética , Linfócitos T/imunologia , Proteínas de Peixe-Zebra/genética , Animais , Códon sem Sentido/genética , Fertilização/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Glutationa/genética , Homozigoto , Humanos , Degradação do RNAm Mediada por Códon sem Sentido/imunologia , RNA Mensageiro/genética , Fatores de Transcrição/genética , Transcriptoma/genética , Peixe-Zebra/genética
20.
Nucleic Acids Res ; 48(13): 7468-7482, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32542372

RESUMO

Alternative polyadenylation (APA) produces transcript 3' untranslated regions (3'UTRs) with distinct sequences, lengths, stabilities and functions. We show here that APA products include a class of cryptic nonsense-mediated mRNA decay (NMD) substrates with extended 3'UTRs that gene- or transcript-level analyses of NMD often fail to detect. Transcriptome-wide, the core NMD factor UPF1 preferentially recognizes long 3'UTR products of APA, leading to their systematic downregulation. Counteracting this mechanism, the multifunctional RNA-binding protein PTBP1 regulates the balance of short and long 3'UTR isoforms by inhibiting NMD, in addition to its previously described modulation of co-transcriptional polyadenylation (polyA) site choice. Further, we find that many transcripts with altered APA isoform abundance across multiple tumor types are controlled by NMD. Together, our findings reveal a widespread role for NMD in shaping the outcomes of APA.


Assuntos
Degradação do RNAm Mediada por Códon sem Sentido , Poliadenilação , Regiões 3' não Traduzidas , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Transativadores/metabolismo , Transcriptoma
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